Soil microbial community response to corrinoids is shaped by a natural reservoir of vitamin B12.

Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

Soil microbial community response to corrinoids is shaped by a natural reservoir of vitamin B12.

Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

Protein binding assays for an accurate differentiation of vitamin B12 from its inactive analogue. A study on edible cricket powder.

Inactive analogues of vitamin B12 (cobalamin, Cbl) can mimic the active Cbl in food if using the traditional microbiological measurements. Thus, overestimated Cbl was recently revealed in edible insects employing immunoaffinity adsorption, HPLC-separation and mass spectrometry (https://doi.org/10.1016/j.foodchem.2021.129048). Here we demonstrate the utility of a convenient binding assay to evaluate Cbl in edible cricket powders. The assay employed the Cbl-specific protein intrinsic factor (IF) and the analogue-detecting protein haptocorrin. The excessive analogues had a weak affinity for IF, resulting in a modest overestimate of Cbl. This overestimate was corrected by a novel mathematical procedure, based on the ratio of analogue/Cbl in the sample and their relative affinities for IF. We found that 100 g of cricket powders contained 40-60 µg of analogues and 0.75-2.2 µg of Cbl. This result was confirmed by HPLC. A correct approach to Cbl-measurements is essential for nutritional assessment of any analogue-containing food.

The inorganic chemistry of the cobalt corrinoids - an update.

The inorganic chemistry of the cobalt corrinoids, derivatives of vitamin B12, is reviewed, with particular emphasis on equilibrium constants for, and kinetics of, their axial ligand substitution reactions. The role the corrin ligand plays in controlling and modifying the properties of the metal ion is emphasised. Other aspects of the chemistry of these compounds, including their structure, corrinoid complexes with metals other than cobalt, the redox chemistry of the cobalt corrinoids and their chemical redox reactions, and their photochemistry are discussed. Their role as catalysts in non-biological reactions and aspects of their organometallic chemistry are briefly mentioned. Particular mention is made of the role that computational methods - and especially DFT calculations - have played in developing our understanding of the inorganic chemistry of these compounds. A brief overview of the biological chemistry of the B12-dependent enzymes is also given for the reader's convenience.

Pseudovitamin B12 and factor S are the predominant corrinoid compounds in edible cricket products.

In this study, we determined the vitamin B12 content of commercially-available edible insect products using a bioassay based on Lactobacillus delbrueckii ATCC 7830. Although the vitamin content of giant water bug, bee larva, grasshopper, and weaver ant products was low, we found that diving beetle and cricket products contained relatively high amounts of vitamin B12 (approximately 89.5 and 65.8 µg/100 g dry weight, respectively). In the cricket products most widely circulated as foods, specific corrinoid (vitamin B12) compounds were extracted and identified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Despite the bioassay detecting high vitamin B12 content (approximately 50-75 µg/100 g dry weight) in these cricket products, UPLC-MS/MS analysis indicated that pseudovitamin B12 and 2-methylmercaptoadenyl cobamide (also known as factor S) were actually the predominant corrinoid compounds (~74% and ~21%, respectively), with authentic vitamin B12 making up only 5% of total corrinoids.

Identification of a Novel Cobamide Remodeling Enzyme in the Beneficial Human Gut Bacterium Akkermansia muciniphila.

The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila strain MucT is known to use cobamides, the vitamin B12 family of cofactors with structural diversity in the lower ligand. However, A. muciniphila MucT is unable to synthesize cobamides de novo, and the specific forms that can be used by A. muciniphila have not been examined. We found that the levels of growth of A. muciniphila MucT were nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that had been studied previously. Unexpectedly, this promiscuity is due to cobamide remodeling-the removal and replacement of the lower ligand-despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share similarity with other cobamide remodeling enzymes or B12-binding domains and is instead a member of the apurinic/apyrimidinic (AP) endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides that they cannot otherwise use in order to grow under cobamide-requiring conditions; this function was confirmed by heterologous expression of cbiR in Escherichia coli Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment.IMPORTANCE Cobamides, comprising the vitamin B12 family of cobalt-containing cofactors, are required for metabolism in all domains of life, including most bacteria. Cobamides have structural variability in the lower ligand, and selectivity for particular cobamides has been observed in most organisms studied to date. Here, we discovered that the beneficial human gut bacterium Akkermansia muciniphila can use a diverse range of cobamides due to its ability to change the cobamide structure via a process termed cobamide remodeling. We identify and characterize the novel enzyme CbiR that is necessary for initiating the cobamide remodeling process. The discovery of this enzyme has implications for understanding the ecological role of A. muciniphila in the gut and the functions of other bacteria that produce this enzyme.

Abiotic reductive deiodination of iodinated organic compounds and X-ray contrast media catalyzed by free corrinoids.

Iodinated X-ray contrast media are known for their stability concerning deiodination in the aquatic environment under aerobic conditions. In this study, we demonstrate the abiotic reductive deiodination of the iodinated contrast media iopromide, iopamidol and diatrizoate in the presence of corrinoids. In addition, triiodinated benzoic acid derivatives with iodine atoms bound at different positions were investigated. Corrinoids like cyanocobalamin (vitamin B12) and dicyanocobinamide served as electron shuttles and as catalysts between the reducing agent (e.g., titanium (III) citrate) and the electron accepting iodinated compound. The concentration decrease of the iodinated compounds followed first-order kinetics with rate constant kobs depending on the iodinated compound. A linear correlation between the rate of iodide release and the corrinoid concentration was observed, with deiodination rates for dicyanocobinamide twice as high as for vitamin B12. Reducing agents with a less negative standard redox potential like dithiothreitol or cysteine caused slower deiodination as the cobalt center was only reduced to its CoII oxidation state. With a temperature increase from 11 to 23 °C, the concentrations of released iodide doubled. A complete deiodination was only observed for the iodinated contrast media but not for structurally similar iodinated benzoic acid derivatives.

A Nickel(II)-Containing Vitamin†B12 Derivative with a Cofactor-F430-type π-System.

F430 is a unique enzymatic cofactor in the production and oxidation of methane by strictly anaerobic bacteria. The key enzyme methyl coenzyme M reductase (MCR) contains a hydroporphinoid nickel complex with a characteristic absorption maximum at around 430 nm in its active site. Herein, the three-step semisynthesis of a hybrid NiII -containing corrinoid that partly resembles F430 in its structural and spectroscopic features from vitamin B12 is presented. A key step of the route is the simultaneous demetalation and ring closure reaction of a 5,6-secocobalamin to metal-free 5,6-dihydroxy-5,6-dihydrohydrogenobalamin with cobaltocene and KCN under reductive conditions. Studies on the coordination chemistry of the novel compound support an earlier hypothesis why nature carefully selected a corphin over a corrin ligand in F430 for challenging nickel-catalyzed biochemical reactions.

Receptor uptake arrays for vitamin B12, siderophores, and glycans shape bacterial communities.

Molecular variants of vitamin B12, siderophores, and glycans occur. To take up variant forms, bacteria may express an array of receptors. The gut microbe Bacteroides thetaiotaomicron has three different receptors to take up variants of vitamin B12 and 88 receptors to take up various glycans. The design of receptor arrays reflects key processes that shape cellular evolution. Competition may focus each species on a subset of the available nutrient diversity. Some gut bacteria can take up only a narrow range of carbohydrates, whereas species such as B. thetaiotaomicron can digest many different complex glycans. Comparison of different nutrients, habitats, and genomes provides opportunity to test hypotheses about the breadth of receptor arrays. Another important process concerns fluctuations in nutrient availability. Such fluctuations enhance the value of cellular sensors, which gain information about environmental availability and adjust receptor deployment. Bacteria often adjust receptor expression in response to fluctuations of particular carbohydrate food sources. Some species may adjust expression of uptake receptors for specific siderophores. How do cells use sensor information to control the response to fluctuations? This question about regulatory wiring relates to problems that arise in control theory and artificial intelligence. Control theory clarifies how to analyze environmental fluctuations in relation to the design of sensors and response systems. Recent advances in deep learning studies of artificial intelligence focus on the architecture of regulatory wiring and the ways in which complex control networks represent and classify environmental states. I emphasize the similar design problems that arise in cellular evolution, control theory, and artificial intelligence. I connect those broad conceptual aspects to many testable hypotheses for bacterial uptake of vitamin B12, siderophores, and glycans.

An Amoebal Grazer of Cyanobacteria Requires Cobalamin Produced by Heterotrophic Bacteria.

Amoebae are unicellular eukaryotes that consume microbial prey through phagocytosis, playing a role in shaping microbial food webs. Many amoebal species can be cultivated axenically in rich media or monoxenically with a single bacterial prey species. Here, we characterize heterolobosean amoeba LPG3, a recent natural isolate, which is unable to grow on unicellular cyanobacteria, its primary food source, in the absence of a heterotrophic bacterium, a Pseudomonas species coisolate. To investigate the molecular basis of this requirement for heterotrophic bacteria, we performed a screen using the defined nonredundant transposon library of Vibrio cholerae, which implicated genes in corrinoid uptake and biosynthesis. Furthermore, cobalamin synthase deletion mutations in V. cholerae and the Pseudomonas species coisolate do not support the growth of amoeba LPG3 on cyanobacteria. While cyanobacteria are robust producers of a corrinoid variant called pseudocobalamin, this variant does not support the growth of amoeba LPG3. Instead, we show that it requires cobalamin that is produced by the Pseudomonas species coisolate. The diversity of eukaryotes utilizing corrinoids is poorly understood, and this amoebal corrinoid auxotroph serves as a model for examining predator-prey interactions and micronutrient transfer in bacterivores underpinning microbial food webs.IMPORTANCE Cyanobacteria are important primary producers in aquatic environments, where they are grazed upon by a variety of phagotrophic protists and, hence, have an impact on nutrient flux at the base of microbial food webs. Here, we characterize amoebal isolate LPG3, which consumes cyanobacteria as its primary food source but also requires heterotrophic bacteria as a source of corrinoid vitamins. Amoeba LPG3 specifically requires the corrinoid variant produced by heterotrophic bacteria and cannot grow on cyanobacteria alone, as they produce a different corrinoid variant. This same corrinoid specificity is also exhibited by other eukaryotes, including humans and algae. This amoebal model system allows us to dissect predator-prey interactions to uncover factors that may shape microbial food webs while also providing insight into corrinoid specificity in eukaryotes.

Decoding molecular interactions in microbial communities.

Microbial communities govern numerous fundamental processes on earth. Discovering and tracking molecular interactions among microbes is critical for understanding how single species and complex communities impact their associated host or natural environment. While recent technological developments in DNA sequencing and functional imaging have led to new and deeper levels of understanding, we are limited now by our inability to predict and interpret the intricate relationships and interspecies dependencies within these communities. In this review, we highlight the multifaceted approaches investigators have taken within their areas of research to decode interspecies molecular interactions that occur between microbes. Understanding these principles can give us greater insight into ecological interactions in natural environments and within synthetic consortia.

Structural basis for universal corrinoid recognition by the cobalamin transport protein haptocorrin.

Cobalamin (Cbl; vitamin B12) is an essential micronutrient synthesized only by bacteria. Mammals have developed a sophisticated uptake system to capture the vitamin from the diet. Cbl transport is mediated by three transport proteins: transcobalamin, intrinsic factor, and haptocorrin (HC). All three proteins have a similar overall structure but a different selectivity for corrinoids. Here, we present the crystal structures of human HC in complex with cyanocobalamin and cobinamide at 2.35 and 3.0 Å resolution, respectively. The structures reveal that many of the interactions with the corrin ring are conserved among the human Cbl transporters. However, the non-conserved residues Asn-120, Arg-357, and Asn-373 form distinct interactions allowing for stabilization of corrinoids other than Cbl. A central binding motif forms interactions with the e- and f-side chains of the corrin ring and is conserved in corrinoid-binding proteins of other species. In addition, the α- and ß-domains of HC form several unique interdomain contacts and have a higher shape complementarity than those of intrinsic factor and transcobalamin. The stabilization of ligands by all of these interactions is reflected in higher melting temperatures of the protein-ligand complexes. Our structural analysis offers fundamental insights into the unique binding behavior of HC and completes the picture of Cbl interaction with its three transport proteins.